Scalable mRNA and siRNA Lipid Nanoparticle Production Using a Parallelized Microfluidic Device.

A major challenge to advance lipid nanoparticles (LNPs) for RNA therapeutics is the development of formulations that can be produced reliably across the various scales of drug development. Microfluidics can generate LNPs with precisely defined properties, but have been limited by challenges in scaling throughput. To address this challenge, we present a scalable, parallelized microfluidic device (PMD) that incorporates an array of 128 mixing channels that operate simultaneously. The PMD achieves a >100× production rate compared to single microfluidic channels, without sacrificing desirable LNP physical properties and potency typical of microfluidic-generated LNPs. In mice, we show superior delivery of LNPs encapsulating either Factor VII siRNA or luciferase-encoding mRNA generated using a PMD compared to conventional mixing, with a 4-fold increase in hepatic gene silencing and 5-fold increase in luciferase expression, respectively. These results suggest that this PMD can generate scalable and reproducible LNP formulations needed for emerging clinical applications, including RNA therapeutics and vaccines.

Towards the Clinical Translation of a Silver Sulfide Nanoparticle Contrast Agent: Large Scale Production with a Highly Parallelized Microfluidic Chip.

Ultrasmall silver sulfide nanoparticles (Ag 2 S-NP) have been identified as promising contrast agents for a number of modalities and in particular for dual-energy mammography. These Ag 2 S-NP have demonstrated marked advantages over clinically available agents with the ability to generate higher contrast with high biocompatibility. However, current synthesis methods are low-throughput and highly time-intensive, limiting the possibility of large animal studies or eventual clinical use of this potential imaging agent. We herein report the use of a scalable silicon microfluidic system (SSMS) for the large-scale synthesis of Ag 2 S-NP. Using SSMS chips with 1 channel, 10 parallelized channels, and 256 parallelized channels, we determined that the Ag 2 S-NP produced were of similar quality as measured by core size, concentration, UV-visible spectrometry, and in vitro contrast generation. Moreover, by combining parallelized chips with increasing reagent concentration, we were able to increase output by an overall factor of 3,400. We also found that in vivo imaging contrast generation was consistent across synthesis methods and confirmed renal clearance of the ultrasmall nanoparticles. Finally, we found best-in-class clearance of the Ag 2 S-NP occurred within 24 hours. These studies have identified a promising method for the large-scale production of Ag 2 S-NP, paving the way for eventual clinical translation.

Scaling up the throughput of microfluidic droplet-based materials synthesis: A review of recent progress and outlook.

The last two decades have witnessed tremendous progress in the development of microfluidic chips that generate micrometer- and nanometer-scale materials. These chips allow precise control over composition, structure, and particle uniformity not achievable using conventional methods. These microfluidic-generated materials have demonstrated enormous potential for applications in medicine, agriculture, food processing, acoustic, and optical meta-materials, and more. However, because the basis of these chips' performance is their precise control of fluid flows at the micrometer scale, their operation is limited to the inherently low throughputs dictated by the physics of multiphasic flows in micro-channels. This limitation on throughput results in material production rates that are too low for most practical applications. In recent years, however, significant progress has been made to tackle this challenge by designing microchip architectures that incorporate multiple microfluidic devices onto single chips. These devices can be operated in parallel to increase throughput while retaining the benefits of microfluidic particle generation. In this review, we will highlight recent work in this area and share our perspective on the key unsolved challenges and opportunities in this field.

Robust Microfabrication of Highly Parallelized Three-Dimensional Microfluidics on Silicon.

We present a new, robust three dimensional microfabrication method for highly parallel microfluidics, to improve the throughput of on-chip material synthesis by allowing parallel and simultaneous operation of many replicate devices on a single chip. Recently, parallelized microfluidic chips fabricated in Silicon and glass have been developed to increase the throughput of microfluidic materials synthesis to an industrially relevant scale. These parallelized microfluidic chips require large arrays (>10,000) of Through Silicon Vias (TSVs) to deliver fluid from delivery channels to the parallelized devices. Ideally, these TSVs should have a small footprint to allow a high density of features to be packed into a single chip, have channels on both sides of the wafer, and at the same time minimize debris generation and wafer warping to enable permanent bonding of the device to glass. Because of these requirements and challenges, previous approaches cannot be easily applied to produce three dimensional microfluidic chips with a large array of TSVs. To address these issues, in this paper we report a fabrication strategy for the robust fabrication of three-dimensional Silicon microfluidic chips consisting of a dense array of TSVs, designed specifically for highly parallelized microfluidics. In particular, we have developed a two-layer TSV design that allows small diameter vias (d < 20 µm) without sacrificing the mechanical stability of the chip and a patterned SiO2 etch-stop layer to replace the use of carrier wafers in Deep Reactive Ion Etching (DRIE). Our microfabrication strategy allows >50,000 (d = 15 µm) TSVs to be fabricated on a single 4" wafer, using only conventional semiconductor fabrication equipment, with 100% yield (M = 16 chips) compared to 30% using previous approaches. We demonstrated the utility of these fabrication strategies by developing a chip that incorporates 20,160 flow focusing droplet generators onto a single 4" Silicon wafer, representing a 100% increase in the total number of droplet generators than previously reported. To demonstrate the utility of this chip for generating pharmaceutical microparticle formulations, we generated 5-9 µm polycaprolactone particles with a CV < 5% at a rate as high as 60 g/hr (>1 trillion particles/hour).

Large-scale production of compound bubbles using parallelized microfluidics for efficient extraction of metal ions.

Correction for 'Large-scale production of compound bubbles using parallelized microfluidics for efficient extraction of metal ions' by Heon-Ho Jeong et al., Lab Chip, 2019, 19, 665-673.

Large-scale production of compound bubbles using parallelized microfluidics for efficient extraction of metal ions.

Recent advances in microfluidic technologies have enabled production of micro-scale compound bubbles that consist of gaseous cores surrounded by thin liquid shells, achieving control and uniformity not possible using conventional techniques. These compound bubbles have demonstrated enormous utility as functional materials for drug delivery, as ultra-lightweight structural materials, as engineered acoustic materials, and also as separating agents for extraction of metal ions from waste fluid streams. Despite these successful demonstrations, compound bubbles have largely remained at the laboratory-scale due to the slow production rates endemic to microfluidics (<10 mL h-1). Although parallelization approaches have enabled large-scale production of simple emulsions and bubbles, its application to the production of higher order dispersions such as compound bubbles has been limited because the optimal processing window for the production of uniform compound bubbles is relatively narrow and the required channel geometry is quite complex. In this report, we demonstrate the parallelization of multi-stage flow focusing droplet generators that produce compound ternary bubbles. We parallelize 400 multi-stage FFG devices, generating up to 3 L (∼1011 bubbles) of monodispersed (CV < 5%) compound bubbles in less than 1 hour. We show that it is critical to use multi-height channels and operate each individual generator in a flow regime that is minimally sensitive to variations in the flow rate to reliably produce uniform compound bubbles. To demonstrate the utility of our parallelized device, we take advantage of the buoyancy and the high mass transfer rate that comes from the thin shells of gas-in-oil-in-water compound bubbles to rapidly extract Nd ions from a model waste stream.

Silicon and glass very large scale microfluidic droplet integration for terascale generation of polymer microparticles.

Microfluidic chips can generate emulsions, which can be used to synthesize polymer microparticles that have superior pharmacological performance compared to particles prepared by conventional techniques. However, low production rates of microfluidics remains a challenge to successfully translate laboratory discoveries to commercial manufacturing. We present a silicon and glass device that incorporates an array of 10,260 (285 × 36) microfluidic droplet generators that uses only a single set of inlets and outlets, increasing throughput by >10,000× compared to microfluidics with a single generator. Our design breaks the tradeoff between the number of generators and the maximum throughput of individual generators by incorporating high aspect ratio flow resistors. We test these design strategies by generating hexadecane microdroplets at >1 trillion droplets per h with a coefficient of variation CV <3%. To demonstrate the synthesis of biocompatible microparticles, we generated 8-16 µm polycaprolactone particles with a CV <5% at a rate of 277 g h-1.

Microfluidic diafiltration-on-chip using an integrated magnetic peristaltic micropump.

Diafiltration is a membrane filtration technique that rapidly removes permeable molecules from a solution by controlling the tangential and orthogonal flows over a membrane and by replenishing the permeate with an equivalent amount of replacement buffer. However, its application to the purification of many key biomaterials and nanomaterials has been limited by the large dead volume (>10 mL) that is required to automate the process. To address this challenge, we have developed a diafiltration-on-a-chip device that can process low-volume samples (50 µL). The key innovation of this device is a magnetically-driven on-chip peristaltic pump that is able to continuously drive fluid flow at rates as high as 50 mL h-1 with minimal external instrumentation and a dead volume of <50 µL. To demonstrate the utility of this device, we purified microbeads from dye with >99% purity and >96% retention within two hours. We additionally showed that cells could be purified from microbeads with >97% purity and >97% retention in two hours. Because our approach requires minimal instrumentation, it is well suited for on-chip parallelization, which was demonstrated by incorporating four complete diafiltration systems onto a single credit card-sized chip.

Liter-scale production of uniform gas bubbles via parallelization of flow-focusing generators.

Microscale gas bubbles have demonstrated enormous utility as versatile templates for the synthesis of functional materials in medicine, ultra-lightweight materials and acoustic metamaterials. In many of these applications, high uniformity of the size of the gas bubbles is critical to achieve the desired properties and functionality. While microfluidics have been used with success to create gas bubbles that have a uniformity not achievable using conventional methods, the inherently low volumetric flow rate of microfluidics has limited its use in most applications. Parallelization of liquid droplet generators, in which many droplet generators are incorporated onto a single chip, has shown great promise for the large scale production of monodisperse liquid emulsion droplets. However, the scale-up of monodisperse gas bubbles using such an approach has remained a challenge because of possible coupling between parallel bubbles generators and feedback effects from the downstream channels. In this report, we systematically investigate the effect of factors such as viscosity of the continuous phase, capillary number, and gas pressure as well as the channel uniformity on the size distribution of gas bubbles in a parallelized microfluidic device. We show that, by optimizing the flow conditions, a device with 400 parallel flow focusing generators on a footprint of 5 × 5 cm2 can be used to generate gas bubbles with a coefficient of variation of less than 5% at a production rate of approximately 1 L h-1. Our results suggest that the optimization of flow conditions using a device with a small number (e.g., 8) of parallel FFGs can facilitate large-scale bubble production.

Ultra-high throughput detection (1 million droplets per second) of fluorescent droplets using a cell phone camera and time domain encoded optofluidics.

Droplet-based assays-in which ultra-sensitive molecular measurements are made by performing millions of parallel experiments in picoliter droplets-have generated enormous enthusiasm due to their single molecule resolution and robustness to reaction conditions. These assays have great untapped potential for point of care diagnostics but are currently confined to laboratory settings due to the instrumentation necessary to serially generate, control, and measure tens of millions of droplets. To address this challenge, we have developed the microdroplet megascale detector (µMD) that can generate and detect the fluorescence of millions of droplets per second (1000× faster than conventional approaches) using only a conventional cell phone camera. The key innovation of our approach is borrowed from the telecommunications industry, wherein we modulate the excitation light with a pseudorandom sequence that enables individual droplets to be resolved that would otherwise overlap due to the limited frame rate of digital cameras. Using this approach, the µMD measures droplets at a rate of 106 droplets per sec (ϕ = 166 mL h-1) in 120 parallel microfluidic channels and achieves a limit of detection LOD = 1 µM Rhodamine dye, sufficient for typical droplet based assays. We incorporate this new droplet detection technology with our previously reported parallelized droplet production strategy, incorporating 200 parallel droplet makers and only one set of continuous and droplet phase inputs and one output line. By miniaturizing and integrating droplet based diagnostics into a handheld format, the µMD platform can translate ultra-sensitive droplet based assays into a self-contained platform for practical use in clinical and industrial settings.

Kilo-scale droplet generation in three-dimensional monolithic elastomer device (3D MED).

Droplet-based microfluidics has led to transformational new approaches in diverse areas including materials synthesis and high-throughput biological assays. However, the translation of droplet microfluidics technology into commercial applications requires scale-up of droplet generation from the laboratory (<10 mL h(-1)) to the industrial (>1 L h(-1)) scale. To address this challenge, we develop a three-dimensional monolithic elastomer device (3D MED) for mass production of monodisperse emulsion droplets. Using double-sided imprinting, 3D microchannels are formed in a single elastomer piece that has 1000 parallel flow focusing generators (k-FFGs). Compared to previous work that parallelizes droplet generation, the 3D MED eliminates the needs for alignment and bonding of multiple pieces and thus makes it possible to achieve the high flow rates and pressure necessary for the kilo-scale generation of droplets. Using this approach, we demonstrate mass production of water-in-oil (W/O) emulsion droplets at production rates as high as 1.5 L h(-1) (>30 billion 45 µm diameter droplets per hour), with a coefficient of variation of droplet diameter of only 6.6%. Because of the simplicity, robustness, and manufacturability of our 3D MED architecture, it is well suited to bridge the gap between the continuously growing library of promising microfluidic technologies to generate microparticles that have been demonstrated in laboratory settings and their successful application in industry.

Ferroplasmons: intense localized surface plasmons in metal-ferromagnetic nanoparticles.

Interaction of photons with matter at length scales far below their wavelengths has given rise to many novel phenomena, including localized surface plasmon resonance (LSPR). However, LSPR with narrow bandwidth (BW) is observed only in a select few noble metals, and ferromagnets are not among them. Here, we report the discovery of LSPR in ferromagnetic Co and CoFe alloy (8% Fe) in contact with Ag in the form of bimetallic nanoparticles prepared by pulsed laser dewetting. These plasmons in metal-ferromagnetic nanostructures, or ferroplasmons (FP) for short, are in the visible spectrum with comparable intensity and BW to those of the LSPRs from the Ag regions. This finding was enabled by electron energy-loss mapping across individual nanoparticles in a monochromated scanning transmission electron microscope. The appearance of the FP is likely due to plasmonic interaction between the contacting Ag and Co nanoparticles. Since there is no previous evidence for materials that simultaneously show ferromagnetism and such intense LSPRs, this discovery may lead to the design of improved plasmonic materials and applications. It also demonstrates that materials with interesting plasmonic properties can be synthesized using bimetallic nanostructures in contact with each other.

Self-organization of nanoscale multilayer liquid metal films: experiment and theory.

Surfaces made from composite nanostructured materials are potential multifunctional platforms for detection, sensing, and energy harvesting in biological and inorganic systems. However, robust and cost-effective synthesis routes are required to create the required arrays of nanostructures with tailorable size, morphology, and composition. Here we show that self-organization via spontaneous pattern formation in nanometer thick bilayer liquid films could lead to such nanostructure arrays. Experimentally, bilayers of immiscible metallic liquids show different self-organized patterning characteristics based on their order of arrangement on a substrate. Energy rate theory based on equating the rate of free energy change to viscous dissipation was used to explain this result. The different bilayer arrangements change the signs of intermolecular interactions, which changes the mode of coupled deformations and the patterning characteristics. Patterning length scale characteristics from nanosecond pulsed laser induced self-organization of Ag and Co liquids on SiO2 substrate were in good agreement with theory.